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. 2023 Mar 21;6:100491. doi: 10.1016/j.crfs.2023.100491

Ginsenoside Rd attenuated hyperglycemia via Akt pathway and modulated gut microbiota in streptozotocin-induced diabetic rats

Wei Wang a, Fengtao Guan b,c, Gianni Sagratini d, Jie Yan b, Jiahan Xie a,c, Zhibo Jin a,c, Meihong Liu a,c, Huimin Liu a,c,∗, Jingsheng Liu a,c,∗∗
PMCID: PMC10074500  PMID: 37033737

Abstract

Ginsenoside Rd is a protopanaxadiol abundant in Panax ginseng and Panax notoginseng. It has been reported that ginsenoside Rd possesses various health benefits, such as anti-diabetic, anti-tumor and anti-inflammatory. This work explored the effects of ginsenoside Rd on hyperglycemia and gut microbiota in streptozotocin-induced diabetic rats. Results showed that 5-week ginsenoside Rd (20 mg/kg) treatment significantly improved hyperglycemia in diabetic rats. Besides, ginsenoside Rd promoted glycogen synthesis via activating Akt pathway. It also inhibited hepatic gluconeogenesis, which was associated with inhibiting phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. We further found that ginsenoside Rd treatment increased the diversity of gut microbiota, increased the abundance of beneficial bacteria, such as SMB53, rc4-4 and Ruminococcus, and reduced the abundance of conditional pathogenic bacteria. These results indicated that ginsenoside Rd has the potential for diabetic intervention.

Keywords: Ginsenoside Rd, Anti-diabetic, Akt pathway, Gut microbiota

Graphical abstract

Image 1

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Highlights

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    Ginsenoside Rd ameliorated hyperglycemia and hepatic pathological damage.

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    Rd inhibited hepatic gluconeogenesis and enhanced hepatic glycogen synthesis.

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    Akt signaling pathway was mediated in the hypoglycemic effect of Rd.

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    Rd modulated gut microbiota composition in hyperglycemia rats.

1. Introduction

According to the International Diabetes Federation (IDF), the number of diabetics is expected to reach 654 million by 2040 (He et al., 2020). Besides, it is estimated that about 90% of diabetic patients are type 2 diabetes mellitus (T2DM). T2DM is a complicated metabolic disease characterized by chronic hyperglycemia (Huang et al., 2021). Long-term hyperglycemia probably triggers chronic complications, including retinopathy and neuropathy (Y. Liu et al., 2019). Recently, anti-hyperglycemic drugs were proven to be poor effects in maintaining stable glycemic, with harmful effects limiting their use (Y. Li et al., 2019). However, natural compounds, including flavonoids, glycosides, and terpenoids, have previously been shown to be effective as medicine in treating diabetes. Therefore, there has been growing interest in natural antidiabetic herbal remedies to treat hyperglycemia.

It is reported that the protein kinase B (Akt) signing pathway was heavily involved in insulin signaling transcription and glucose homeostasis (Seo et al., 2019). Glycogen synthesis kinase-3β (GSK3β) is an essential substrate of Akt, and Akt negatively regulates its activity. Besides, Akt prevents GSK3β from interacting with glycogen synthase (GS) to promote hepatic glycogen synthesis, reducing glucose (Manning and Toker, 2017). In addition, Forkhead box protein O1 (FoxO1) is the most critical downstream nuclear transcription factor of Akt. The action of FoxO1 on phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) is inhibited by phosphorylated Akt (F. Wu et al., 2021). It inhibits hepatic gluconeogenesis to attenuate hyperglycemia.

Gut microbiota is vital to human health and is an indispensable symbiont for the human body. Recently, accumulated evidence indicates a strong association between gut microbiota dysbiosis and T2DM, such as an increase in the proportion of Bacteroides/Firmicutes, as well as the increase of various opportunistic pathogens, and the increase of producing short-chain fatty acids bacteria (Zheng et al., 2021). Various natural medicines were reported to reverse gut microbiota dysbiosis in diabetic rats (Ryuk et al., 2017; Xu et al., 2021; Zhao et al., 2020). For example, Berberis extract alleviates glucose metabolism in T2DM rats by regulating gut microbiota composition (Ryuk et al., 2017). However, whether the ginsenoside Rd can alleviate hyperglycemia by the alteration of gut microbiota composition in T2DM rats is still unknown.

Ginsenoside Rd is a tetracyclic triterpene derivative with white powder, mainly found in ginseng and Panax ginseng (Z. Liu et al., 2022). Numerous studies indicated that ginsenoside Rd possessed many biological activities, such as neurological protection, anti-cancer, and anti-tumor effects (Ren et al., 2021; Sun et al., 2011; You et al., 2021). Chu et al. reported that ginsenoside Rd could play a neuroprotective role in neurons in the diabetic state (Chu et al., 2014). In addition, Tang et al. reported that ginsenoside Rd could restore diabetic hyperglycemia-induced endothelial cell loss and other retinal damage by activating AMPK and SIRT1 in vivo (Tang et al., 2022). However, ginsenoside Rd was rarely reported to regulate glucose metabolism based on the Akt signaling pathway in T2DM rats. In this study, we explained the possible function of ginsenoside Rd on regulating hepatic glucose metabolism via the Akt pathway and regulating gut microbiota homeostasis to attenuate hyperglycemia. Our results will provide some theoretical knowledge in the prevention and treatment of T2DM.

2. Materials and methods

2.1. Animals and treatment

Male SD rats (180 ± 20 g,8 weeks old) were purchased from (Beijing Vital River Experimental Animal Co., Ltd, Beijing, China). All rats were acclimated under a controlled environment (20 ± 2 °C, 12 h light/dark cycle), free to drink and eat. Besides, the normal control (NC) group received a standard diet, and other groups received a high-fat and high-sugar diet (77% rat basal maintenance diet + 10% lard + 5% egg yolk powder + 5% sucrose + 2.5% cholesterol + 0.5% bile acid sodium). All experiments were performed following the recommendations in the national guidelines “Regulations for the Administration of Affairs Concerning Experimental Animals” and were approved by the USA Committee of the Jilin Institute of Traditional Chinese Medicine (SYXK (JI) 2015-0009).

The SD rats were fed for 4 weeks, fasted for 12 h, and the normal diet group was injected with 0.01 mol/L citrate buffer (pH = 4), and the other groups were injected with 40 mg/kg streptozotocin (1%, pH = 4). Rats with >16.7 mmol/L blood glucose 3 days later but still >16.7 mmol/L within 1 week were defined as hyperglycemic rats. And then, SD rats were divided into four groups based on the mean blood glucose, which were NC group (n = 12), model control (MC) group (n = 15), metformin (Met) group (n = 15), ginsenoside Rd low-dose (Rd-10) group (n = 15), and ginsenoside Rd high-dose (Rd-20) group (n = 15). NC group and MC group were given sodium carboxymethyl cellulose solution. Besides, another group of rats treated with low-dose and high-dose ginsenoside Rd and metformin was given 10, 20 and 300 mg/kg daily intragastric administration, respectively. During the five-week intervention, weight was measured daily, and blood glucose was monitored every week. In addition, the rats fasted for 12 h, and FBG was measured again before death.

2.2. Oral glucose tolerance tests and insulin tolerance tests

Oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) were conducted at 8 and 9 weeks. Before being intraperitoneally injected with either glucose (2 g/kg body weight) or insulin (1 U/kg body weight), the basal blood glucose levels of the rats in each group were measured. Additional levels were tested at 30, 60, 90, and 120 min. The area under the curve (AUC) calculations were used to determine the experimental results.

2.3. Detection of biochemical index

Blood samples were collected in heparin-coated tubes and centrifuged (4 °C, 3000 rpm) to obtain serum. The serum indicators of high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (TC), triacylglycerol (TG), aspartate transaminase (AST) and alanine aminotransferase (ALT) were detected using commercial detection kits (Shanghai Yuanye Biotechnology Co., LT, Shanghai, China).

2.4. Histopathological analysis

Liver and pancreatic tissue were removed from physiological vials containing paraformaldehyde and trimmed. They were rinsed with running water for 5 h. In addition, the trimmed tissues were sequentially subjected to ethanol gradient dehydration, xylene washing, wax dipping, sectioning (5 μm), and baking. Sections were stained with hematoxylin and eosin, observing morphology.

2.5. Real-time quantitative PCR analysis

Total RNA was extracted from liver tissue using Trizol reagent (Takara, Dalian, China). When genomic DNA in total RNA was removed, complementary cDNA was synthesized using a Prime Script RT reagent kit (Takara, Japan), and the resulting cDNA was placed in a −20 °C freezer for storage. Once the real-time fluorescence PCR reaction mix had been configured, the cDNA was amplified by PCP according to the appropriate procedure. The specific primers used are shown in Table S1.

2.6. Western blotting analysis

Total proteins in liver tissue were extracted by grinding beads with RIPA lysate, and then the target proteins were obtained by centrifugation. Equal amounts of them were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with the corresponding primary antibodies overnight before membranes were probed with corresponding secondary antibodies. Densitometry was performed with the Image J Software and normalized to β-actin.

2.7. High-throughput sequencing of fecal 16sRNA in T2DM rats

The QIAamp Fast DNA Stool Mini Kit was used to extract the rat fecal colony DNA. Using a NanoDrop device, DNA concentration and purity were assessed after DNA extraction was finished. PCR amplified the V3-V4 region of the 16S rRNA gene. The Illumina MiSeq platform and the MiSeq Reagent Kit v3 were then used by Shanghai Personal Biotechnology Co., Ltd. to undertake paired-end 2 × 250 bp sequencing.

2.8. Statistical analysis

Statistical analysis was performed using Prism 9.0.0 GraphPad software. All measurement data were presented in mean ± standard deviation (SD). Comparisons of statistical significance between groups were determined using Student's t-test or one-way analysis of variance (ANOVA) followed by Duncan's multiple tests. P < 0.05 was considered statistically significant.

3. Results

3.1. Effects of ginsenoside Rd on body weight and FBG in T2DM rats

To assess whether ginsenoside Rd can improve the phenotype of hyperglycemia and emaciation in T2DM, body weight and FBG were measured regularly each week. As shown in Fig. 1A, high-fat and high-sugar diet pretreatment led to body weight gain during the rats modeling period over time. Moreover, compared to the MC group, the body weight in Rd-10 and Rd-20 groups was significantly enhanced with the treatment of ginsenoside Rd (P < 0.01). From Fig. 1B, after intraperitoneal injection of streptozotocin, the FBG in the MC group and therapy groups exhibited a significant increase from the seventh to tenth week compared to the NC group (P < 0.01). Notably, FBG levels of the Rd-10 group showed no remarkable decrease compared to the MC group, while the Rd-20 group showed an opposite trend. In a word, ginsenoside Rd (20 mg/kg) reversed the symptoms of body weight loss and hyperglycemia.

Fig. 1.

Fig. 1

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Effect of ginsenoside Rd on body weight and blood glucose in diabetic rats. (A) Body weight of rats (n = 8). (B) Fasting blood glucose of rats (n = 8). (C) OGTT experiment: blood glucose at 0, 30, 60, 90, and 120 min. (D) AUC of OGTT data. (E) ITT experiment: blood glucose at 0, 30, 60, 90 and 120 min. (F) AUC of ITT data. Data are expressed as mean ± SD (n = 6). #P < 0.05 and ##P < 0.01 vs. NC group; *P < 0.05 and **P < 0.01 vs. MC group.

3.2. Effects of ginsenoside Rd on OGTT and ITT in T2DM rats

To explore the function of ginsenoside Rd in blood glucose control, OGTT and ITT were performed during the study period. The blood glucose level in the MC group was consistently higher than that of the treatment groups after 30 min. Furthermore, the AUC value in the MC group reached 3.8 times that of the NC group (Fig. 1D). The ITT results revealed a significantly lower rate of blood glucose decline in the T2DM groups than that of the rats in the NC group (P < 0.01), which suggests that insulin sensitivity of diabetic rats has deteriorated seriously. However, ginsenoside Rd (20 mg/kg) significantly improved the feedback to insulin in T2DM rats compared with the MC group, which was reflected in lower AUC values in the Rd-20 group (Fig. 1F). In a word, ginsenoside Rd improved impaired glucose and insulin tolerance in a dose-dependent manner in T2DM rats.

3.3. Effects of ginsenoside Rd on lipid profiles in the serum and liver in T2DM rats

T2DM usually is associated with impaired liver function and dyslipidemia. Therefore, related indicators are tested to evaluate whether ginsenoside Rd could improve liver function and blood lipids. As shown in Table 1, TG, TC and LDL levels were significantly higher than the NC group, indicating dyslipidemia in the T2DM rats. However, this unnormal trend could be reversed by the intervention of ginsenoside Rd.

Table 1.

Effects of ginsenoside Rd on serum lipid profile.

Group TC/(mmol/L) TG/(mmol/L) LDL/(mmol/L) HDL/(mmol/L) AST/(U/L) ALT/(U/L)
NC 0.17 ± 0.06** 0.70 ± 0.26** 0.60 ± 0.09** 1.70 ± 0.23** 71.20 ± 9.15** 29.11 ± 6.34**
MC 4.32 ± 0.46 10.21 ± 1.83 2.68 ± 0.55 0.83 ± 0.16 107.36 ± 24.86 89.78 ± 5.04
Met 0.46 ± 0.16** 1.80 ± 1.64** 0.92 ± 0.21** 1.27 ± 0.37* 75.43 ± 8.62** 35.33 ± 5.16**
Rd-10 1.06 ± 0.45** 1.89 ± 0.94** 1.91 ± 0.38** 1.75 ± 0.33** 69.69 ± 7.92** 60.08 ± 14.45**
Rd-20 0.41 ± 0.20** 0.86 ± 0.63** 1.20 ± 0.47** 1.60 ± 0.33** 59.07 ± 7.01** 25.32 ± 11.50**
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TC: total cholesterol, TG: triacylglycerol, LDL: low-density lipoprotein, HDL: high-density lipoprotein, AST: aspartate aminotransferase, ALT: alanine aminotransferase. Data are expressed as mean ± SD (n = 8). *P < 0.05 and **P < 0.01 vs. MC group.

AST and ALT are the most critical indicators of liver function, whose levels reflect the changes in liver function. As shown in Table 1, the Rd-20 group had the lowest AST and ALT levels, significantly different from the MC group. In addition, the hepatocytes in the MC group rats were heterogeneous in size, and there was notable steatosis and swelling, along with the emergence of many vesicles (Fig. 2B). Consistent with the AST and ALT results, histopathological analysis of the liver showed that the diseased liver tissue was progressing towards normalization with the treatment of ginsenoside Rd. According to the above results, it is evident that the normalization of blood lipids and liver function can be promoted by ginsenoside Rd, especially at 20 mg/kg.

Fig. 2.

Fig. 2

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Effect of ginsenoside Rd on pathological changes in the liver and pancreas of diabetic rats. (A) Representative photomicrographs of H&E stained liver tissues. (B) Representative photomicrographs of H&E stained pancreatic tissues. Scale bar = 100 μm, number of samples (n = 6).

3.4. Effects of ginsenoside Rd on hepatic gluconeogenesis and glycogen synthesis in T2DM rats

Gluconeogenesis and glycogen synthesis are effective ways for organisms to regulate blood glucose. PEPCK and G6pase are the rate-limiting enzymes in the hepatic gluconeogenesis process. Besides, GS is a key enzyme involved in glycogen synthesis and is the substrate of GSK3β. Hence, the mRNA and protein expression levels of PEPCK, G6pase, GSK3β and GS were measured by q-PCR and western blot. As shown in Fig. 3A, PEPCK and G6pase were up-regulated in the MC group but inhibited by ginsenoside Rd treatment. Glycogen synthesis was suppressed in T2DM rats, specifically manifested at a decrease in GSK3β phosphorylation levels and an increase in GS phosphorylation levels. However, ginsenoside Rd could reverse this situation by increasing GS expression and inhibiting GSK3β expression.

Fig. 3.

Fig. 3

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Expression analysis of glycogen synthesis, gluconeogenesis-related genes (A) and Akt pathway-related genes (B) in the liver. Relative mRNA expression levels were detected by RT-qPCR. Data are expressed as mean ± SD (n = 3). *P < 0.05 and **P < 0.01 vs. MC group.

It can be seen from Fig. 4 that the expression levels of G6pase, PEPCK and p-GS proteins in the MC group presented an increasing trend than other groups, while the expression levels of p-GSK3β protein decreased. Meanwhile, we discovered that ginsenoside Rd reversed changes in these critical proteins. The level of p-GSK3β protein significantly improved and the level of p-GS was prominently decreased in ginsenoside Rd treatment groups (P < 0.01). The above results suggest that ginsenoside Rd can regulate hepatic gluconeogenesis and glycogen synthesis processes, especially at 20 mg/kg.

Fig. 4.

Fig. 4

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Effects of ginsenoside Rd on hepatic glycogen synthesis, gluconeogenesis and Akt signaling pathway protein expression. Expression protein levels of p-GSK3β, GSK3β, G6pase, PEPCK, p-GS, GS (A) and p-Akt, Akt, p-FoxO1, FoxO1, GLUT2 (B) and p-GSK3β, GSK3β, G6pase, PEPCK, p-GS, GS relative protein levels (C) and p-Akt, Akt, p-FoxO1, FoxO1, GLUT2 relative protein levels (D) are analyzed by western blot analysis. Data are expressed as mean ± SD (n = 3). *P < 0.05 and **P < 0.01 vs. MC group.

3.5. Effects of ginsenoside Rd on Akt mRNA and protein signaling pathway in T2DM rats

Activation of the Akt signaling pathway is closely associated with hepatic gluconeogenesis and glycogen synthesis. Therefore, we examined the change in crucial gene and protein levels under the ginsenoside Rd intervention. As indicated in Fig. 3B, the expression level of FoxO1 was significantly decreased under the treatment of Rd. In contrast, the expression level of Akt was reversed (P < 0.01). Likewise, Glucose transporter type 2 (GLUT2) plays a unique role as a glucose transporter protein for hepatocytes during glucose metabolism in the liver. We found that GLUT2 was notably up-regulated by ginsenoside Rd treatment from Fig. 3B (P < 0.01).

The expression of Akt pathway-related proteins, including Akt, FoxO1 and GLUT2, were analyzed by Western Blot. From Fig. 4C, the expression level of phosphorylated FoxO1 (p-FoxO1) protein was significantly increased under the treatment of Rd (20 mg/kg). Compared with untreated T2DM rats, the p-Akt and GLUT2 protein levels in the Rd-20 group were significantly increased 2.23 fold and 2.44 fold, respectively (P < 0.01). In a word, Rd treatment significantly regulated FoxO1 transcription and GLUT2 activity in a dose-dependent manner, thereby interfering with hepatic glucose metabolism.

3.6. Effects of ginsenoside Rd on gut microbiota composition in T2DM rats

Evidence of varying degrees supports that gut microbiota exerts an essential role in human health. Therefore, we investigated the improvement effect of ginsenoside Rd on the disturbed gut microbiota by 16s rRNA high-throughput sequencing technology. As shown in Fig. 5A–D, microbiota diversity and taxonomic composition in T2DM rats were enhanced by the ginsenoside Rd intervention (20 mg/kg). In addition, PCA analysis revealed a noticeable difference in gut microbiota between the MC group and NC group (Fig. 6A). The microbiota composition at the genus level displayed the abundance of Lactobacillus and Clostridium in the MC group was about 60%. However, their abundance decreased significantly under treatment with ginsenoside Rd (Fig. 5E). Further analysis revealed that the main contributor of Lactobacillus in the MC group was Lactobacillus-helveticus (Fig. 5F). Notably, both metformin and ginsenoside Rd treatment reversed this trend, reflected in lower Lactobacillus-helveticus levels (Fig. 5K).

Fig. 5.

Fig. 5

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Effects of ginsenoside Rd on the change of gut microbiota composition in T2DM rats. Alpha diversity indices: Chao1 (A), Pielou (B), Shannon (C) and Simpson (D). The relative abundance of top 10 at the bacterial genus levels (E). The relative abundance of top 10 at the bacterial species levels (F). Quantitative relative abundance of the gut microbiota: Enterococcus (G), SMB53 (H), rc4-4 (I), [Ruminococcus] (J), Lactobacillus-helveticus (K), Clostridium-celatum (L). Data are expressed as mean ± SD (n = 5). *P < 0.05 and **P < 0.01 vs. MC group.

Fig. 6.

Fig. 6

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Changes in gut microbiota composition with ginsenoside Rd treatment are associated with hepatic glucose metabolism in T2DM rats. (A) Beta diversity and principal component analysis. (B) LDA analysis. (C) Heat map of the relationship between gut microbiota and physical-biochemical indicators in different groups. Number of samples (n = 5). *P < 0.05 and **P < 0.01 vs. MC group.

LDA analysis was used to identify statistically significant enrichment taxa in different groups. From Fig. 6B, Turicibacterales, Turicibacteraceae and Turicibacters were considered as representative gut microbiota of Rd-10 group. In addition, Bacteroidetes, including its members Bacteroidia and Bacteroidetes, were increased significantly in the MC group. Notably, SMB53 was identified as the only abundant genus in the Rd-20 group. Spearman's correlation analysis was performed to explore the relationships between microbiota composition alteration and hyperglycemia-related indices. As depicted in Fig. 6C, Lactobacillus-helveticus, Clostridium-celatum and Turicibacter abundance were significantly positively correlated with OGTT, ITT and FBG. However, Oscillospira, Desulfovibrionales, rc4-4 and Enterococcus showed negative correlations with OGTT, ITT and FBG, indicating their intensive association with blood glucose improvement. Briefly, the anti-diabetic effect of ginsenoside Rd is related to the improvement of gut microbiota structure.

4. Discussion

Hyperglycemia is one of the manifestations of T2DM, managed with synthetic drugs, including metformin and insulin injections. However, prolonged usage of synthetic medications has been shown to have hazardous adverse effects. Various active ingredients of medicinal plants were extracted to replace synthetic drugs (Zhang et al., 2017). Ginsenosides are the main active components in Panax notoginseng and Panax ginseng, which can be divided into many types according to the different glycoside structures. It is reported that ginsenoside Rg5 improved glucolipid metabolism and insulin resistance in diabetic mice (Wei et al., 2020). Besides, Liu et al. demonstrated that ginsenoside Rk3 could alleviate insulin resistance and improve gluconeogenesis in T2DM mice (Y. Liu et al., 2019). Similarly, we found that ginsenoside Rd (20 mg/kg) significantly alleviated hyperglycemia and hyperlipidemia in T2DM rats. And it is evidenced by the decrease in FBG, TC, TG, and LDL levels and the increase in HDL levels. The hypoglycemic effect of ginsenoside Rd was proven by the glucose and insulin tolerance improvement in T2DM rats (Fig. 1).

Besides, we found that the hypoglycemic effect of ginsenoside Rd on T2DM rats was not remarkable as the hypolipidemic effect. In this study, there was a significant difference between T2DM rats of ginsenoside Rd treatment (10 mg/kg or 20 mg/kg) and normal rats in blood glucose levels. In addition, several clinical trials showed that ginsenosides preparations are not very effective in lowering glucose in T2DM patients, which may be related to the degree of diabetes, body weight, and physical effort of the patients (Jovanovski et al., 2014; Oh et al., 2014). Moreover, we also assumed this result may be caused by ginsenoside Rd dose and T2DM animal model selection. However, further evidence in vivo is still needed. Specifically, different T2DM animal models should be applied to evaluate the effect of attenuating hyperglycemia from ginsenoside Rd.

The liver is the primary place for the metabolism, synthesis and redistribution of glucose, where hepatic gluconeogenesis and glycogen synthesis are two crucial means involved in regulating glucose (C. Wu et al., 2005). The synthesis of glucose into glycogen is called glycogen synthesis, where GS is the main rate-limiting enzyme and GSK3β can regulate this metabolic process through its own activity at the level of phosphorylation and dephosphorylation. T2DM leads to down-regulation of GS expression and up-regulation of GSK3β expression. Our study found that ginsenoside Rd significantly increased p-GSK3β expression and prevented GSK3β from binding to GS, thereby accelerating hepatic glycogen synthesis in T2DM rats (Fig. 3). In addition, the occurrence of hyperglycemia in T2DM is partially attributed to the acceleration of hepatic glucose production by activation of G6Pase and PEPCK. Our study found that ginsenoside Rd inhibited the expression levels of G6pase and PEPCK, thereby decreasing hepatic gluconeogenesis. In a word, these results indicate that ginsenoside Rd attenuates hyperglycemia by reducing hepatic gluconeogenesis and increasing glycogen synthesis.

Akt is a key factor in regulating hepatic glycogen synthesis and hepatic gluconeogenesis in the insulin signaling pathway (Wei et al., 2020). Related studies have shown that hyperglycemia in T2DM is attributed to the blockage of the Akt signaling pathway, which prevents the downstream factor FoxO1 from exerting its transcriptional effects (JiTing et al., 2018; Q. Liu et al., 2017). The consequence is the inhibition of GS and the enhancement of G6pase and PEPCK. Besides, Fan et al. indicated that ginsenoside Rg1 could reduce glucose output from HepG2 cells by increasing phosphorylation of Akt and decreasing GSK3β expression (Fan et al., 2019). Meanwhile, our study showed that ginsenoside Rd enhanced the activity of p-FoxO1, p-GSK3β and GLUT2 in T2DM rats by activating Akt, thereby inhibiting the enhanced G6pase and PEPCK activities and increasing GS activities (Fig. 7). Therefore, the above results point to a mechanism of ginsenoside Rd based on the Akt signaling pathway to interfere with hyperglycemia in T2DM that may be attributed to the following. On the one hand, Akt/FoxO1-mediated inhibition of PEPCK and G6pase expression contributed to reducing hepatic gluconeogenesis. On the other hand, Akt/GSK3β-mediated GS activation increased hepatic glycogen synthesis.

Fig. 7.

Fig. 7

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Schematic illustration of the molecular mechanism by which ginsenoside Rd improves high-sugar and high-fat diet/streptozotocin-induced T2DM in rats.

It has been reported that changes in the gut microbiota structure are closely related to T2DM phenotypes, including hyperglycemia and hyperlipidemia (Arora et al., 2021). The research on new targets based on gut microbiota for preventing T2DM is gradually progressing. Numerous studies have shown that ginsenosides can improve gut microbiota imbalance (Long et al., 2022). Currently, we found that ginsenoside Rd could also regulate gut microbiota composition in T2DM rats. Specifically, Lactobacillus-helveticus and Clostridium-celatum were significantly decreased with ginsenoside Rd treatment, which was strongly associated with weight loss and glycemic abnormalities. Mainly, SMB5S was the only microbiota marker in the Rd-20 group. Besides, the abundance of SMB53, rc4-4 and Ruminococcus was significantly increased with the intervention of ginsenoside Rd (20 mg/kg), which were negatively correlated with hyperglycemia by Spearman's correlation analysis, suggesting that ginsenoside Rd can modulate the abundance of beneficial bacteria to alleviate T2DM. Meanwhile, related studies have further demonstrated that some genera, including SMB53, rc4-4 and Ruminococcus, can produce short-chain fatty acids, which can participate in the gluconeogenesis pathway and thus regulate glucose homeostasis (Z.-R. Li et al., 2021; G. Liu et al., 2018). Therefore, the findings of this study suggest that ginsenoside Rd may be able to prevent gut microbiota dysbiosis in T2DM rats.

5. Conclusion

In conclusion, ginsenoside Rd effectively ameliorated hyperglycemia in streptozotocin-induced T2DM rats. The anti-diabetic effect of ginsenoside might be related to increased hepatic glycogen synthesis and decreased gluconeogenesis via enhancement of the Akt signaling pathway. Moreover, ginsenoside Rd treatment ameliorated hyperglycemia probably through the modulation of gut microbiota composition. In future study, the effect of ginsenoside Rd on hyperglycemia also needs to be verified through clinical population trials. Our findings highlight that ginsenoside Rd has the potential to develop healthy foods and prevent diabetes.

CRediT authorship contribution statement

Wei Wang: Validation, Methodology, Data curation, Software. Fengtao Guan: Data curation, Formal analysis. Gianni Sagratini: Conceptualization, Writing – review & editing. Jie Yan: Methodology, Data curation. Jiahan Xie: Methodology, Data curation. Zhibo Jin: Methodology, Data curation, Formal analysis. Meihong Liu: Writing – review & editing. Huimin Liu: Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Writing - review & editing. Jingsheng Liu: Validation, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This research was funded by National Key R&D Program of China (2022YFF1100205), Research Project Between Universita’ Degli Studi Di Camerino And Jilin Agricultural University, the China Agriculture Research System of MOF and MARA.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.crfs.2023.100491.

Contributor Information

Gianni Sagratini, Email: gianni.sagratini@unicam.it.

Huimin Liu, Email: liuhuimin@jlau.edu.cn.

Jingsheng Liu, Email: liujingsheng@jlau.edu.cn.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (20.3KB, docx)

Data availability

No data was used for the research described in the article.

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